Blood coagulation is a process consisting of a complex interaction of various blood components (or factors) that gives rise to a fibrin clot. Generally, the blood components, which participate in what has been referred to as the coagulation “cascade”, are enzymatically inactive proteins (proenzymes or zymogens) that are converted to proteolytic enzymes by the action of an activator (which itself is an activated clotting factor). Coagulation factors that have undergone such a conversion are generally referred to as “active factors”, and are designated by the addition of the letter “a” to the name of the coagulation factor (e. g. Factor XIIIa).
FXIII is predominantly found in the zymogen form in either recombinant FXIII (FXIII-A2), plasma FXIII (FXIII-A2B2) or intracellular FXIII (FXIII-A2). In plasma, during blood clotting, the thrombin activation pathway predominates. Thrombin cleaves an activation peptide of 37 amino acid residues from the N-terminal part of each FXIII-A subunit and in presence of Ca2+ generates the active form FXIIIa*. Conversely, intracellular FXIII is not accessible to thrombin and it has been observed that non-proteolytic activation occurs under certain conditions to yield an active FXIII species, (FXIIIaO) which contains all 1-731 amino acids in each FXIII-A subunit and thus has not been cleaved by activated thrombin (Polgar et al (1990) Biochem. J. 267, 557-560).
For patients with severe hemophilia, blood coagulation factors, such as FXIII are administered to aid in the blood clotting process. FXIII is to be administrated in its in-active/zymogen form, which first will be naturally activated in vivo on demand, e.g when a laceration occurs. As a quality parameter of the product the content of pre-activated FXIII (FXIIIaO) needs to be measured and kept to a minimum. However, there is a likelihood that certain amounts of FXIII may be non-proteolytically activated to FXIIIaO within a pharmaceutical formulation which would consequently increase the risk of non-specific coagulation.
There is thus a great need for a method of measuring the quality of a FXIII containing formulation to ensure the safety of FXIII products and to ensure that treatment of hemophilia is optimised.